Various publications, including patents, published applications and scholarly articles, cited throughout the present specification are incorporated by reference herein, in its entirety. Citations not fully set forth within the specification may be found at the end of the specification.
Sucrose plays an important role in the ultimate aroma and flavor that is delivered by a coffee grain or bean. Sucrose is a major contributor to the total free reducing sugars in coffee, and reducing sugars are important flavor precursors in coffee. During the roasting of coffee grain, reducing sugars will react with amino group containing molecules in a Maillard type reaction, which generates a significant number of products with caramel, sweet and burnt-type aromas and dark colors that are typically associated with coffee flavor (Russwurm, 1969; Holscher and Steinhart, 1995; Badoud, 2000). The highest quality Arabica grain (Coffea Arabica) have been found to have appreciably higher levels of sucrose (between 7.3 and 11.4%) than the lowest quality Robusta grain (Coffea canephora) (between 4 and 5%) (Russwurm, 1969; Illy and Viani, 1995; Chahan et al., 2002; Badoud, 2000). Despite being significantly degraded during roasting, sucrose still remains in the roasted grain at concentrations of 0.4-2.8% dry weight (DW); thereby, contributing directly to coffee sweetness. A clear correlation exists between the level of sucrose in the grain and coffee flavor. Therefore, identifying and isolating the major enzymes responsible for sucrose metabolism and the underlying genetic basis for variations in sucrose metabolism will enable advances in the art of improving coffee quality.
Currently, there are no published reports on the genes or enzymes involved in sucrose metabolism in coffee. However, sucrose metabolism has been studied in tomato Lycopersicon esculentum (a close relative of coffee, both are members of asterid I class), especially during tomato fruit development. An overview of the enzymes directly involved in sucrose metabolism in tomato is shown in FIG. 1 (Nguyen-Quoc et al., 2001). The key reactions in this pathway are (1) the continuous rapid degradation of sucrose in the cytosol by sucrose synthase (SuSy) and cytoplasmic invertase (I), (2) sucrose synthesis by SuSy or sucrose-phosphate synthase (SPS), (3) sucrose hydrolysis in the vacuole or in the apoplast (region external to the plasma membrane, including cell walls, xylem vessels, etc) by acid invertase (vacuolar or cell wall bound) and, (4) the rapid synthesis and breakdown of starch in the amyloplast.
As in other sink organs, the pattern of sucrose unloading is not constant during tomato fruit development. At the early stages of fruit development, sucrose is unloaded intact from the phloem by the symplast pathway (direct connections between cells) and is not degraded to its composite hexoses during unloading. Both the expression and enzyme activity of SuSy are highest at this stage and are directly correlated with sucrose unloading capacity from the phloem (phenomena also called sink strength; Sun, et al., 1992; Zrenner et al., 1995). Later in fruit development, the symplastic connections are lost. Under these conditions of unloading, sucrose is rapidly hydrolyzed outside the fruit cells by the cell wall bound invertase and then the glucose and fructose products are imported into the cells by hexose transporters. Sucrose is subsequently synthesized de novo in the cytoplasm by SuSy or SPS (FIG. 1). SPS catalyses an essentially irreversible reaction in vivo due to its close association with the enzyme sucrose phosphate phosphatase (Echeverria et al., 1997). In parallel to the loss of the symplastic connections, SuSy activity decreases, and eventually becomes undetectable in fruit at the onset of ripening (Robinson et al. 1998; Wang et al. 1993). Therefore, late in the development of tomato fruit, the SPS enzyme, in association with SP, appears as the major enzymes for sucrose synthesis.
Plant invertases have been separated into two groups based on the optimum pH for activity. Invertases of the first group are identified as neutral invertases, which are characterized as having a pH optima in the range of 7-8.5. The neutral invertases have been found to be located in the cytosol of plant cells. Invertases of the second group are identified as acid invertases, which are characterized as having a pH optima for activity between pH 4.5 and 5.5. The acid invertase have been shown to exist in both soluble and insoluble forms (Sturm and Chrispeels, 1990). Insoluble acid invertase is irreversibly and covalently associated with the cell wall; whereas, soluble acid invertase is located in both the vacuole and apoplast.
Research over the past decade has shown that vacuolar as well as cell-wall bound invertase are key enzymes in the regulation of sucrose metabolism during fruit development of various species. Red-fruit species of tomato, such as the commercial species Lycopersicon esculent and the wild species L. pimpinellifolium, for example, do not store high levels of sucrose but, instead, accumulate hexoses in the form of glucose and fructose. Evidence from crosses of red-fruit species with sucrose-accumulating green-fruit species (Yelle et al., 1991) has shown the crucial role of acid invertase in preventing final sucrose accumulation in red-fruited tomato species. Genetic analysis studies have located the locus conferring high levels of soluble solids in L. pimpinellifolium fruit to the known position of vacuolar invertase TIV1 (Tanksley et al., 1996; Grandillo and Tanksley, 1996). A similar conclusion was reached from the analysis of expression of an antisense TIV1 cDNA construction in transgenic tomatoes (Klann et al, 1993; Klann et al., 1996). Thus the vacuolar form of invertase is considered to play a major role in both the regulation of hexose levels in mature fruits and in the regulation of mobilization of sucrose stored in the vacuoles (Klann et al., 1993; Yau and Simon, 2003). The cell wall bound isoforms are believed to be involved in phloem unloading and sucrose partitioning (Scholes et al, 1996).
The importance of cell wall bound invertase has been demonstrated by studies with transgenic tomato (Dickinson et al., 1991) and tobacco (von Schaewen et al., 1990) plants that overexpress cell wall invertase in a constitutive fashion. Elevated levels of invertase activity in such plants caused reduced levels of sucrose transport between sink and source tissues, which lead to stunted growth and overall altered plant morphology. Reduction of extracellular invertase activity has also been shown to have dramatic effects on plant and seed development in various species. Analysis of transgenic carrots with reduced levels of cell wall invertase due to the constitutive expression of an antisense cell wall invertase construct (Tang et al., 1999) has shown dramatic consequences on early plant development as well as on tap root formation during early elongation phase.
Studies of the miniature-1 (mn1) (Lowe and Nelson, 1946) seed mutant in maize, which is characterized by an aberrant pedicel and a drastic reduction in the size of the endosperm, have shown that Mn1 seed locus encodes a cell wall invertase, CWI-2 (Miller and Chourey, 1992; Cheng et al.; 1996). Interestingly, in the mn1 mutant, global acid invertase (vacuolar and cell wall bound) activity is dramatically reduced suggesting coordinate control of both the vacuolar and cell wall enzyme activities.
Because of the importance of sucrose for high quality coffee flavor, a need exists to determine the metabolism of sucrose beans and the interaction of genes involved in that metabolism. There is also a need to identify and isolate the genes that encode these enzymes in coffee, thereby providing genetic and biochemical tools for modifying sucrose production in coffee beans to manipulate the flavor and aroma of the coffee.